Mining And Functional Verification Of Genes Related To Distyly In Primula Forbesii

Distyly is a scientific issue which is related to the reproductive evolution of plants that has puzzled botanists for more than one hundred years.At present,it is well known that distyly is controlled by genes at S locus,however,the regulatory mechanism has not been determined.As a typical distyly plant,Primul forbesii is an ideal material to investigate the formation mechanism of distyly.In the early stage of our research group,we sequenced the transcriptome of Primula forbesii,and got 28 genes differentially expressed between pistils of long style morph(Pin)and short style morph(Thrum).On this basis,genes with significantly different expression between pin and thrum pistils were selected as candidate genes by fluorescence quantitative PCR technique.In order to verify the function of these genes,the virus-induced gene silencing system and genetic transformation system were constructed in Primula forbesii.The functions of candidate genes were verified from the forward and reverse directions,and the key genes related to distyly were mined.The main results are as follows:(1)13 genes were selected from 28 differentially expressed genes as candidate genes by fluorescence quantitative PCR technique,including 8 genes with specific high expression in pin pistils,2 genes with significantly higher expression in pin pistils than that in thrum pistils and 3genes with significantly higher expression in thrum pistils than that in pin pistils.(2)The infection liquid with OD600 value of 1.0 was injected into the plant by the way of leaf back injection,and the infection efficiency was 60%.It was the best VIGS system for the functional verification of the genes in the plant.By using this system to verify the candidate genes,we found that the five high expression genes of the pin pistils,which are auxin transporter gene Pf PIN5,pollen allergen protein gene Pf POE1;4,GDSL lipase gene Pf GELP10,cysteine protease gene Pf RD21 A and polygalacturonase gene Pf PGA4,are closely related to the style length.(3)The overexpression vectors of three genes PfPIN5,PfGELP10 and PfPOE1;4 were successfully constructed and transferred into tobacco.Among them,one of the transgenic positive plants with Pf PIN5 gene has significantly longer style than that of the wild type and the empty vector group.It needs to be further proved that this is caused by gene overexpression.(4)It was first time found that pollen-tube pathway method can be used in gene transformation in long homostyle P.forbesii to verify the function of gene overexpression,which laid the foundation for verifying the function of distyly gene in P.forbesii.Overall,five key genes were found to control the length of style,which may affect the cell elongation and ultimately lead to the change of style length.The results provided new informations for understanding the molecular mechanism of distyly.